Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

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Contributed equally

Abstract

In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.

KEYWORDS:

Identification and in vitro validation of two anti-CRISPR protein families. (A) Schematic of candidate anti-CRISPR proteins and aca2 genes in the genomic context of H. parainfluenzae (AcrIIC4Hpa) and S. muelleri (AcrIIC5Smu). Gray genes are associated with mobile DNA, and known gene functions are annotated as follows: “Reg” is a transcriptional regulator, “Tail” is involved in phage tail morphogenesis, and “Tra” is a transposase. The B. oedipodis aca2 gene is used as a query for pBLAST searches, and percent identities of aca2 orthologs are denoted. Arrows are not drawn to scale. (B) In vitro cleavage of target DNA by the NmeCas9-sgRNA complex in the presence of anti-CRISPR protein. Preformed NmeCas9-sgRNA RNP complex was incubated with purified anti-CRISPR proteins as indicated with AcrE2 as a negative control, AcrIIC1 as a positive control, and candidate Acrs. Then, a linearized plasmid with a protospacer and PAM sequence was added to the reaction mixture. Molarities of anti-CRISPR protein (relative to constant Cas9 molarity) are shown at the top of each lane, mobilities of input and cleaved DNAs are denoted on the right, and cleavage efficiencies (“% cleaved”) are given at the bottom of each lane.

Validation of Cas9 and anti-CRISPR proteins from H. parainfluenzae and S. muelleri. (A) Validation of HpaCas9 and SmuCas9 cleavage activity and inhibition by anti-CRISPR proteins in vitro. The double asterisk denotes sgRNA. (B) Interaction between Acrs and NmeCas9, HpaCas9, and SmuCas9 is visualized by Coomassie blue staining after copurification of each 6×His-tagged Cas9 and untagged Acrs from E. coli. Each Cas9 ortholog and anti-CRISPRs are indicated as arrowheads and asterisks, respectively. (C) Plaquing of E. coli phage Mu targeted by the Nme, Hpa, Cje, or Geo Cas9 in the presence of the anti-CRISPR proteins. Tenfold serial dilutions of phage Mu lysate were spotted on lawns of bacteria expressing the indicated Acr proteins. Data are from one plate representative of ≥3 replicates.

AcrIIC4Hpa and AcrIIC5Smu inhibit genome editing by NmeCas9 in human cells. (A) T7E1 assays of NmeCas9 or SpyCas9 editing efficiencies at two dual target sites (DTS3 and DTS7) upon transient plasmid transfection of human HEK293T cells. Constructs encoding anti-CRISPR proteins were cotransfected as indicated at the top of each lane. Mobilities of edited and unedited bands are indicated to the right, and editing efficiencies (“% lesion”) are given at the bottom of each lane. The figure is a representative of three replicates. (B) A bar graph of editing efficiencies measured by TIDE analysis upon RNP delivery of NmeCas9-sgRNA and Acr into HEK293T cells. Statistical significance was determined by two-tailed paired Student’s t test. Means and standard deviations from three biological replicates are indicated with lines (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

AcrIIC4Hpa and AcrIIC5Smu prevent stable DNA binding by NmeCas9. (A and B) A native gel of the sgRNA visualized by SYBR gold staining (A) and of the FAM-labeled target DNA (B), both of which were added last to NmeCas9 + Acr (and in panel B, + sgRNA) incubation. (C) Live-cell fluorescence images of U2OS cells transiently transfected with plasmids encoding dNmeCas9-(sfGFP)3, dSpyCas9-(mCherry)3, their respective telomeric sgRNAs, and Acrs. The plasmid encoding the Acr is also marked with the blue fluorescent protein, mTagBFP2, which is overlaid on a differential interference contrast (DIC) image of each cell. The specific version of each plasmid set (with or without sgRNAs, with or without anti-CRISPRs) is given to the right of each row. First column, differential interference contrast (DIC) and mTagBFP2 imaging, overlay. Second column, dNmeCas9-(sfGFP)3. Third column, dSpyCas9-(mCherry)3. Fourth column, dNmeCas9-(sfGFP)3 and dSpyCas9-(mCherry)3, merged. Scale bars, 5 µm.

Summary of type II-C Cas9 orthologs and anti-CRISPR families. Type II-C anti-CRISPRs can act at distinct stages of Cas9-mediated target DNA cleavage. While AcrIIC1 binds to the HNH domain and inhibits a broad spectrum of Cas9 orthologs, AcrIIC4 and AcrIIC5 prevent DNA binding and have a narrower range of inhibition, similar to AcrIIC3.